Stabilization and isolation of extracellular nucleic acids

Abstract

The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using an apoptosis inhibitor, preferably a caspase inhibitor, a hypertonic agent and/or a compound according to formula 1 as defined in the claims.

The invention claimed is: 
     
       1. A method for stabilizing an extracellular nucleic acid population comprised in a cell-containing biological sample, comprising:
 contacting a cell-containing biological sample that comprises extracellular nucleic acids with 
 (a) a caspase inhibitor, and 
 (b) at least one compound according to formula 1, 
 
       
         
           
           
               
               
           
         
         
           wherein R1 is a hydrogen residue or an alkyl residue, R2 and R3 are identical or different hydrocarbon residues with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen residue, 
         
         thereby stabilizing an extracellular nucleic acid population comprises in the cell-containing biological sample and providing a stabilized cell-containing biological sample. 
       
     
     
       2. The method according to  claim 1 , wherein a release of genomic DNA from cells contained in the cell-containing biological sample into a cell-free portion of the cell-containing biological sample is reduced and/or a degradation of nucleic acids present in the cell-containing biological sample is reduced due to the stabilization. 
     
     
       3. The method according to  claim 1 , wherein
 a) the caspase inhibitor has one or more of the following characteristics:
 i) it is a pancaspase inhibitor; and/or 
 ii) it comprises a modified caspase-specific peptide; and/or 
 
 b) the compound according to formula 1 has one or more of the following characteristics:
 i) R1 is a C1-C5 alkyl residue; 
 ii) R1, R2 and R3 comprise 1 to 5 carbon atoms; and/or 
 iii) it is a N,N-dialkyl-carboxylic acid amide. 
 
 
     
     
       4. The method according to  claim 1 , wherein after the cell-containing biological sample has been contacted with the caspase inhibitor and the compound according to formula 1, the resulting mixture has one or more of the following characteristics:
 a) it comprises the caspase inhibitor in a concentration selected from at least 0.01 μM, at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at least 3.5 μM; 
 b) it comprises the caspase inhibitor in a concentration range selected from 0.01 μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM or 2.504 to 25 μM; 
 c) it comprises the compound according to formula 1 in a concentration of at least 0.1%, at least 0.5%, at least 1%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%; and/or 
 d) it comprises the compound according to formula 1 in a concentration range selected from 0.1% to 50%, 0.5% to 25%, 0.75% to 20%, 1% to 15% or 1% to 10%. 
 
     
     
       5. The method according to  claim 1 , wherein the cell-containing biological sample is for stabilization contacted with:
 a) at least one caspase inhibitor comprising a modified caspase-specific peptide as caspase inhibitor, and 
 b) at least one N,N-dialkyl-carboxylic acid amide, 
 optionally wherein the compounds according to a) and b) are comprised in a stabilising composition. 
 
     
     
       6. The method according to  claim 1 , wherein the cell-containing biological sample is a body fluid. 
     
     
       7. The method according to  claim 6 , wherein the body fluid is selected from whole blood, plasma and/or serum. 
     
     
       8. The method according to  claim 1 , wherein stabilization of the extracellular nucleic acid population is achieved without refrigeration for a time period selected from
 a) at least two days; 
 b) at least three days; 
 c) at least one day to three days; 
 d) at least one day to six days; and/or 
 e) at least one day to seven days. 
 
     
     
       9. The method according to  claim 1 ,
 wherein the caspase inhibitor, the at least one compound according to formula 1 and optionally further additives are comprised in a stabilising composition, and 
 wherein the volumetric ratio of the stabilising composition to the specified volume of the cell-containing biological sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. 
 
     
     
       10. The method according to  claim 1 , further comprising analyzing and/or processing extracellular nucleic acids of the stabilized cell-containing biological sample. 
     
     
       11. The method according to  claim 10 , comprising removing cells from the stabilized cell-containing biological sample and isolating extracellular nucleic acids from the remaining sample from which cells were removed. 
     
     
       12. The method according to  claim 11 , wherein nucleic acids are isolated from cells that were removed from the stabilized cell-containing biological sample. 
     
     
       13. The method according to  claim 10 , wherein the cell-containing biological sample has one or more of the following characteristics:
 i) it is a body fluid; and/or 
 ii) it is selected from whole blood, plasma and/or serum. 
 
     
     
       14. A method for isolating extracellular nucleic acids from a cell-containing biological sample comprising:
 a) contacting a cell-containing biological sample that comprises extracellular nucleic acids with
 (a) a caspase inhibitor, and 
 (b) at least one compound according to formula 1, 
 
 
       
         
           
           
               
               
           
         
         
           
             wherein R1 is a hydrogen residue or an alkyl residue, R2 and R3 are identical or different hydrocarbon residues with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and R4 is an oxygen residue, 
           
           thereby stabilizing an extracellular nucleic acid population comprised in the cell-containing biological sample and providing a stabilized cell-containing biological sample; and 
         
         b) isolating extracellular nucleic acids from the stabilized cell-containing biological sample. 
       
     
     
       15. The method according to  claim 14 , wherein
 a) the caspase inhibitor has one or more of the following characteristics:
 i) it is a pancaspase inhibitor; and/or 
 ii) it comprises a modified caspase-specific peptide; and/or 
 
 b) the compound according to formula 1 has one or more of the following characteristics:
 i) R1 is a C1-05 alkyl residue; 
 ii) R1, R2 and R3 comprise 1 to 5 carbon atoms; and/or 
 iii) it is a N,N-dialkyl-carboxylic acid amide. 
 
 
     
     
       16. The method according to  claim 14 , wherein the cell-containing biological sample has one or more of the following characteristics:
 i) it is a body fluid; and/or 
 ii) it is selected from whole blood, plasma and/or serum. 
 
     
     
       17. The method according to  claim 14 , comprising one or more of the following steps:
 i) optionally removing cells from the cell-containing biological sample between step a) and step b); 
 ii) performing one or more of the following steps:
 aa) the stabilized sample is subjected to a nucleic acid analysis and/or detection method; 
 bb) extracellular nucleic acids are isolated from the stabilized sample; 
 cc) extracellular nucleic acids are isolated from the stabilized sample and the isolated nucleic acids are analysed and/or detected; 
 dd) cells comprised in the stabilized sample are removed; 
 ee) cells comprised in the stabilized sample are removed prior to performing an isolation, analysis and/or detection step; 
 ff) a nucleic acid isolation step is performed after a stabilization period of at least two days, at least three days, at least one day to three days, at least one day to six days, and/or at least one day to seven days; 
 gg) (i) the stabilized sample, (ii) the stabilized sample from which cells have been removed and/or (iii) cells removed from the sample are stored; 
 hh) cells that were removed from the stabilized sample are discarded; and/or 
 ii) nucleic acids are isolated from cells that were removed from the stabilized sample; 
 
 and/or 
 iii) step b) is performed using an isolation method selected from the group comprising extraction, solid-phase extraction, isolation methods using a nucleic acid binding solid phase, isolation methods using a silica material, isolation methods that are based on the use of a solid phase comprising anionic exchange groups; magnetic particle-based purification, phenol-chloroform extraction, alcohol and/or chaotropic agent(s) based nucleic isolation method, chromatography, anion-exchange chromatography, anion exchange particle-based isolation, electrophoresis, filtration, precipitation, target nucleic acid specific isolation methods and combinations thereof. 
 
     
     
       18. The method according to  claim 14 , wherein the isolated nucleic acids are in a further step c) processed and/or analyzed. 
     
     
       19. The method according to  claim 14 , wherein
 a) the extracellular nucleic acid population that is isolated from the cell-free portion of the sample and/or that is obtained after isolation in step b) has one or more of the following characteristics:
 i) it is comprised as a portion in the total nucleic acid that is isolated; 
 ii) it predominantly comprises DNA; 
 iii) it predominantly comprises RNA; 
 iv) it comprises circulating extracellular nucleic acids; 
 v) it comprises disease related nucleic acids; 
 vi) it comprises tumor-associated or tumor-derived nucleic acids; 
 vii) it comprises inflammation related nucleic acids: 
 viii) it comprises fetal nucleic acids; 
 ix) it comprises viral nucleic acids; 
 x) it comprises pathogen nucleic acids; 
 xi) it comprises mammalian extracellular nucleic acids; and/or 
 xii) it is a mixture of DNA and RNA; 
 
 and/or 
 b) the extracellular nucleic acid that is analyzed and/or further processed in step c), has one or more of the following characteristics:
 i) it is DNA; 
 ii) it is RNA; 
 iii) it is a circulating extracellular nucleic acid; 
 iv) it comprises disease related nucleic acids; 
 v) it comprises tumor-associated or tumor-derived nucleic acids; 
 vi) it comprises inflammation related nucleic acids: 
 vii) it is a fetal nucleic acid; 
 viii) it is a viral nucleic acid; 
 ix) it is a pathogen nucleic acid; 
 x) it is a mammalian extracellular nucleic acid; and/or 
 xi) it is a mixture of DNA and RNA. 
 
 
     
     
       20. The method according to  claim 1 , wherein the cell-containing biological sample is additionally contacted with at least one anticoagulant and/or at least one hypertonic agent which stabilizes the cells comprised in the sample. 
     
     
       21. The method according to  claim 20 , wherein
 (i) the anticoagulant has one or more of the following characteristics:
 it is a chelating agent; and/or 
 it is EDTA; 
 
 and/or 
 (ii) the hypertonic agent has one or more of the following characteristics:
 it is uncharged; 
 it stabilizes the cells comprised in the sample by inducing cell shrinking; 
 it is cell impermeable; 
 it is water-soluble; 
 it is a hydroxylated organic compound; 
 it is a polyol; 
 it is a hydroxy-carbonyl compound; 
 it is a carbohydrate or a sugar alcohol; and/or 
 it is dihydroxyacetone. 
 
 
     
     
       22. The method according to  claim 20 , wherein after the cell-containing biological sample has been contacted with the at least one anticoagulant and/or the at least one hypertonic agent, the resulting mixture has one or more of the following characteristics:
 it comprises the hypertonic agent in a concentration of at least 0.05M, at least 0.1 M, at least 0.25M, or at least 0.5M; 
 it comprises the hypertonic agent in a concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M, and 0.1M to 0.6M; and/or 
 it comprises the anticoagulant in a concentration range selected from 0.05 mM to 100 mM, 0.05 mM to 50 mM, 0.1 mM to 30 mM, 1 mM to 20 mM, and 2 mM to 15 mM. 
 
     
     
       23. The method according to  claim 3 , wherein:
 a) the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and/or 
 b) the compound according to formula 1 is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide, or is N,N-dimethylpropanamide. 
 
     
     
       24. The method according to  claim 14 , wherein step a) additionally comprises contacting the cell-containing biological sample with at least one anticoagulant and/or at least one hypertonic agent which stabilizes the cells comprised in the sample. 
     
     
       25. The method according to  claim 24 , wherein
 (i) the anticoagulant has one or more of the following characteristics:
 it is a chelating agent; and/or 
 it is EDTA; 
 
 and/or 
 (ii) the hypertonic agent has one or more of the following characteristics:
 it is uncharged; 
 it stabilizes the cells comprised in the sample by inducing cell shrinking; 
 it is cell impermeable; 
 it is water-soluble; 
 it is a hydroxylated organic compound; 
 it is a polyol; 
 it is a hydroxy-carbonyl compound; 
 it is a carbohydrate or a sugar alcohol; and/or 
 it is dihydroxyacetone. 
 
 
     
     
       26. The method according to  claim 24 , wherein after the cell-containing biological sample has been contacted with the at least one anticoagulant and/or the at least one hypertonic agent, the resulting mixture has one or more of the following characteristics:
 it comprises the hypertonic agent in a concentration of at least 0.05M, at least 0.1 M, at least 0.25M, or at least 0.5M; 
 it comprises the hypertonic agent in a concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M, and 0.1M to 0.6M; and/or 
 it comprises the anticoagulant in a concentration range selected from 0.05 mM to 100 mM, 0.05 mM to 50 mM, 0.1 mM to 30 mM, 1 mM to 20 mM, and 2 mM to 15 mM. 
 
     
     
       27. The method according to  claim 15 , wherein:
 a) the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK; and/or 
 b) the compound according to formula 1 is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide, or is N,N-dimethylpropanamide.